Project 1: Molecular physiology and regulation of volume-sensitive chloride channels Cells respond to swelling by activating anion and cation channels that allow passive loss of inorganic ions and water, a response known as regulatory volume decrease (RVD), which is an important cellular homeostatic mechanism for maintaining cell volume. Volume-sensitive chloride (C1-) channels are ubiquitously present in mammalian cells, including heart and vascular cells, and play an important physiological role in a variety of other diverse cellular functions as well, including the regulation of electrical activity, intracellular pH, immunological responses, cell proliferation and differentiation. The actual cascade of intracellular events which links cell volume changes to the regulation of volume-sensitive C1-channels is presently unknown, and the identification of the protein responsible for these channels has remained elusive. The P.I. and his collaborators have recently identified ClC-3, a member of the ClC superfamily of voltage-dependent Cl-channels, as a strong molecular candidate responsible for volume-sensitive Cl-channels in heart and Cl-channels. The objectives of this project include: (1) an examination of the role of several regulatory proteins and other ClC family members on the properties of volume-sensitive Cl-channels in native cardiac cells, and recombinant ClC-3 Cl- channels expressed in NIH 3T3 cells, (2) characterize the intracellular signaling pathways which link changes in cell volume to the activation of volume-sensitive Cl- channels in native cells and expressed recombinant Clc-3 Cl- channels, volume using site-directed mutagenesis, deletions and chimeric ClC-3 channels. Since the activation of cardiac Cl-channels can produce significant effects on action potential duration and automaticity, and are key regulators of cell volume homeostasis, these channels have important clinical significance for several myocardial diseases, including cardiac arrhythmias, myocardial ischemia, congestive heart failure and hypertrophy. This project has significant potential of elucidating the normal physiological and possible pathophysiological role of volume- sensitive Cl-channels in the heart and cardiovascular system.